The long-term objective of this proposal is to establish a unified theory to describe the mechanisms of crosstalk signaling between Ca2+ and reactive oxygen species (ROS) in cardiac muscle cells, and to translate these signaling mechanisms to the physiology and pathology of cardiac function. The pivotal role of mitochondrial Ca2+ and ROS in mediating the life and death of cardiac muscle cells is well recognized. The separation between mitochondria-mediated life versus death resides in the finest balance between concentrations of Ca2+ and ROS. The majority of existing research in the field focuses individually either on Ca2+ or ROS homeostasis. The interaction between these two signaling pathways, however, has just begun to gain attention by a small number of laboratories. Intriguingly, there is a growing library of literature suggesting that mitochondrial dynamics (fission, fusion, and trafficking) play an essential role in the physiological regulation of cellular ATP, Ca2+, and ROS homeostasis. In this proposal, we will study how these three important components (Ca2+, ROS, and mitochondrial fission machinery) communicate to regulate cardiac Ca2+ and ROS crosstalk signaling. We will use a multidisciplinary approach encompassing techniques of cell biology (e.g. confocal microscopy), molecular biology (e.g. gene transfer), biochemistry (e.g. western blots), and transgenic mouse models (e.g. cyclophilin D (CypD) knockout mice and mt-cpYFP transgenic mice) to elucidate the mechanisms of ROS and Ca2+ symbiosis with an unique emphasis on mitochondrial fission protein DLP1 and mitochondrial permeability transition (MPT). Our central hypothesis is: an increased mitochondrial Ca2+ concentration ([Ca2+]m) favors the balance of mitochondrial dynamics towards fission that in turn increases ROS generation. The resulting oxidized environment leads to additional mitochondrial Ca2+ influx. Both the increases in [Ca2+]m and ROS enhance the opening probability of MPT that further augments ROS generation. Eventually, this positive feedback loop is counter balanced by Ca2+ and ROS activated mitochondrial Ca2+ efflux mechanisms including Na/Ca exchange and MPT. The three specific aims are: 1) To determine whether an increased [Ca2+]m promotes mitochondrial fission processes, which then lead to increase ROS generation. 2) To assess the contribution of a CypD containing MPT pathway in [Ca2+]m-mediated ROS generation. 3) To determine the role of MPT as a rapid Ca2+ efflux mechanism of mitochondria. PUBLIC HEALTH RELEVANCE: Oxidative stress can cause numerous human diseases including cardiomyopathy in chronic heart failure, ischemic heart disease, neurodegenerative diseases, diabetes, obesity, and aging. The proposed research focuses on the elucidation of cellular mechanisms of reactive oxygen species generation. It is our objective, not only to make a scientific contribution to oxidative stress-mediated disease mechanisms, but also to develop possible therapeutic means for treating these debilitating disorders.